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Biomedical subjects

Ning Guo

Publications and source records attributed to Ning Guo.

31 records · Page 2Linked to original sources
PubMed2004

A new format of bispecific antibody: highly efficient heterodimerization, expression and tumor cell lysis.

Bispecific antibodies (BsAbs) have been considered as potential therapeutics for cancer. A major obstacle in the development of BsAb has been the difficulty in producing a heterodimer with two different arms and in sufficient quantity for clinical application by the traditional methods. We describe a new format of BsAb that consists of two single-chain variable fragment of antibodies (scFvs), one for human epidermal growth factor receptor 2 (HER2)/neu and the other for CD16, heterodimerized by a "knobs-into-holes" device from the CH3 domains of the human IgG1 Fc fragment. The two chains were functionally expressed in CHO cells and assembled into heterodimers with dual antigen-binding specificity. Compared with other types of engineered BsAbs expressed in mammalian cells, the yield of this BsAb was relatively high (12-14 mg/l). In vitro experiments demonstrated that the BsAb was able to recruit human peripheral blood mononuclear cells (PBMC) to kill SK-BR-3 cells more effectively than the commercial anti-HER2/neu antibody Herceptin (Roche, Shanghai). This new format of BsAb possesses properties that support its potential as a new antitumor agent.

Animals↗
PubMed2004

Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene.

To increase the selection efficiency and productivity of stable clones expressing recombinant antibody-IL-2 fusion protein, a dicistronic expression vector containing the anti-erbB2 scFv-Fc-IL-2 fusion gene followed by a weakened neo gene, was constructed to allow for the concurrent translation of the recombinant protein and mutated neomycin phosphotransferase from a single mRNA. The presence of the mutant enzyme in the transfectomas resulted in a decreased resistance of cells in the presence of an elevated level of G418 and retarded cell growth. The transfectomas containing the mutant enzyme expressed considerably higher levels of the fusion protein than those containing the normal enzyme. Furthermore, these positive clones had an almost identical level of recombinant gene expression, which was very stable even when the concentration of G418 was significantly increased. Thus, the selection efficiency of strongly positive producers was remarkably increased. Our results demonstrate that the dicistronic expression vector is useful for the selection of highly expressing clones. Combined with an amplification system, this vector may have potential usage for the expression of recombinant antibody and the yield may be further improved by this expression system.

Animals↗
PubMed2004

The difference of binding epitopes on human CD16 (FcgammaRIII) interacted with hIgG1 and monoclonal antibody B88-9.

The 3-D complex structures of extracellular domain of human CD16 (FcgammaRIII) and hIgG1 or B88-9 were obtained by means of computer-guided molecular modeling, respectively. The binding epitopes were predicted and binding energy was calculated theoretically. The epitopes of human CD16 interacted with hIgG1 and B88-9 were different and the binding energy of B88-9 was stronger than hIgG1. The computer competing simulation results showed that B88-9 could bind to CD16 when CD16 was also bound by hIgG1, while IgG1 could not bind to CD16 after CD16 was bound by B88-9. By flow cytometry analysis, the competitive binding activity of B88-9 to CD16 with hIgG1 was evaluated. With the increasing concentrations of hIgG1, the binding activity of B88-9 was not interfered markedly. It suggested that hIgG1 could not inhibit the interaction of CD16 and B88-9 and the binding domain of hIgG1 and B88-9 on CD16 was different.

Antibodies, Monoclonal↗
PubMed2004

[Mannosylation of ligand binding domain 2 of HER-2/neu].

AIM: To establish a new method for protein mannosylation, which will facilitate the study of antigen presentation mediated by mannose receptor. METHODS: Recombinant HER-2/neu ligand binding domain 2 (LBD2) was purified through DEAE sepharose FF column and cobalt-based affinity resins. LBD2 protein was mannosylated in-vitro,and then analyzed by MALDI-TOF-MS and resorcinol-sulfuric acid method. RESULTS: The purity of LBD2 obtained was 90%. Analysis of MALDI-TOF-MS and resorcinol-sulfuric acid method suggested LBD2 was mannosylated. CONCLUSION: LBD2 neoglycoprotein has been obtained, which lays the foundation for the study of antigen uptake and presentation mediated by mannose receptor and the development of tumor vaccine.

Binding Sites↗
PubMed2003

A trivalent anti-erbB2/anti-CD16 bispecific antibody retargeting NK cells against human breast cancer cells.

Bispecific antibody (BsAb) can physically cross-link immune cells to tumor cells, circumventing the proper structures for tumor cell-immune cell interactions and activating the cellular cytotoxic mechanisms. The optimal BsAb should target tumor cells with high affinity, but activate trigger molecules on cytotoxic cells by monovalent binding of Fab fragments. In the present study, a trivalent anti-erbB2/anti-CD16 BsAb was produced. This BsAb possesses bivalent arms specifically binding to the extracellular domain of erbB2 and monovalent Fab fragment redirecting NK cells. The recombinant protein could be expressed and purified from Escherichia coli as native proteins without refolding. It was fully functional in bispecific binding to SKBR3 and NK cells. The molecular size of this trivalent BsAb protein is larger than diabody and smaller than whole antibody and expected to have advantages for both high penetration of small antibody fragments and the slow circulation clearance of whole antibody. This novel protein may be an attractive target for further improvement and evaluation.

Antibodies, Bispecific↗
PubMed2003

Design and assembly of anti-CD16 ScFv antibody with two different linker peptides.

Several studies have focused on the effect of different lengths of linker peptides on the properties of single-chain Fv (ScFv). The expressing level and stability of anti-CD16 ScFv with common linker peptide (Gly(4)Ser)(3) were very poor. Considering 3-D structures of heavy and light chain variable region gene of anti-CD16 antibody, a novel linker peptide PT7 (i.e. Gly(3)SerAla(3)) was designed. As a comparison, the linker PT5 (Gly(4)Ser) was chosen to construct anti-CD16 ScFv. A molecular modeling of anti-CD16 ScFv antibody with the two different linker peptides was designed using computer-assisted modeling techniques and molecular dynamics method. Based on the crystal structure of human IgG1 Fc fragment-Fc gamma receptor III (Fc gamma RIII) complex, putative interactions between anti-CD16 ScFv antibody with two different linker peptide and Fc gamma receptor III fragment were predicted with Docking method. Using molecular graphic techniques, the structure-function relationship of anti-CD16 ScFv antibody with two different linker peptides was analyzed and the combining ability was predicted. The binding activity to Daudi cells by FACS showed that anti-CD16 ScFv antibody with two different linker peptides possessed similar ability and the experimental result was consistent with the theoretical prediction.

Drug Design↗
PubMed2003

The role of insulin-like growth factor-II in cancer growth and progression evidenced by the use of ribozymes and prostate cancer progression models.

Towards understanding the IGF system during cancer growth and progression, progressive prostate cancer models, such as SV40 large T antigen immortalized human prostate epithelial cells (P69, M2182, M2205, and M12) and LNCaP sublines (C4, C4-2, and C4-2B4), were used. IGF-II mRNA levels progressively increase as prostate cancer cells become more tumorigenic and metastatic, suggesting that IGF-II contributes in part to prostate cancer progression. The role of IGF-II in cancer cell growth was evaluated in LNCaP, PC3, and M12 prostate cancer cell lines and MCF-7 breast cancer cell line by ribozyme/antisense strategies which were previously shown to suppress endogenous IGF-II expression and cell growth in PC-3 cells [Xu et al., Endocrinol 140 (1999) 2134]. Retroviral mediated transient expression of IGF-II-specific ribozyme (RZ) caused extensive cell death. In stably cloned cell lines, both RZ and mutant ribozyme (MRZ) inhibited cancer cell growth, suggesting that antisense effects of MRZ may be sufficient for cell growth inhibition. These results confirm an important role of IGF-II in cancer cell growth and progression, and support further development of gene therapy targeting IGF-II.

Animals↗
PubMed2003

A recombinant anti-erbB2, scFv-Fc-IL-2 fusion protein retains antigen specificity and cytokine function.

Elevated erbB2 expression is detected in many in situ and invasive human ductal carcinomas. Anti-erbB2 antibody directed at the extracellular domain of erbB2 can result in an antitumor response in some patients with tumors overexpressing erbB2 oncoprotein. By combining interleukin 2 (IL-2) activities with a tumor specific antibody, immunotherapy of tumors might be more effective in the future. In this study, a fusion protein consisting of erbB2 single chain antibody (scFv), Fc fragment of human IgG1 and IL-2 was constructed. The molecular weight of fusion proteins is 66 kDa, only one third of whole antibody-IL-2 fusion protein or 44% whole Ig molecule. The fusion proteins retained the activities of both antigen binding and IL-2. The scFv-Fc-IL-2 fusion protein may have advantages over whole antibody-IL-2 fusion proteins, such as smaller molecule, better activity of penetration, more favorable pharmacokinetic properties.

Animals↗
PubMed2003

[Expression and Characterization of Single-chain Fv-Fc Fusion Protein against Human P185(erbB2)].

In order to increase therapeutic effects and decrease immunogenicity of mouse McAb, the single-chain Fv (scFv) created by fusing the light and heavy chain variable region genes of anti-human P185(erbB2) McAb was conjugated to the Fc gene of human IgG1 to construct a scFv-Fc fusion gene. The scFv-Fc fusion gene was cloned into the expression vector pCIDN. The scFv-Fc fusion protein was synthesized as secreted two-chain molecule in CHO cells, and purified by affinity chromatography on recombinant protein A. A special 185 kD P185(erbB2) protein was immunoprecipitated by the scFv-Fc fusion protein. The fluorescence-activated cell sorting (FACS) using SK-BR-3 cells as the target indicated that the fusion protein could bind to the extracellular domain of P185(erbB2). The affinity of the scFv-Fc fusion protein, determined by ELISA, was K=7.5x10(-10) (mol/L)(-1). This work laid basis for further studies on the anti-P185(erbB2) scFv-Fc fusion protein.

Animals↗
PubMed2002

Carcinogen-induced S-phase arrest is Chk1 mediated and caffeine sensitive.

We have investigated the mechanism of S-phase arrest elicited by the carcinogen benzo(a)pyrene dihydrodiol epoxide (BPDE) in p53-deficient cells. Inhibition of DNA synthesis after BPDE treatment was rapid and dose dependent (approximately 50% inhibition after 2 h with 50 nM BPDE). Cells treated with low doses (50-100 nM) of BPDE resumed DNA synthesis after a delay of approximately 4-8 h, whereas cells that received high doses of BPDE (600 nM) failed to recover from S-phase arrest. The checkpoint kinase Chk1 (but not Chk2) was phosphorylated after treatment with low doses of BPDE. High concentrations of BPDE elicited phosphorylation of both Chk1 and Chk2. Adenovirus-mediated expression of "dominant-negative" Chk1 (but not dominant-negative Chk2) and the Chk1 inhibitor UCN-01 abrogated the S-phase delay elicited by low doses of BPDE. Consistent with a role for the caffeine-sensitive ATM or ATR protein kinase in low-dose BPDE-induced S-phase arrest, both Chk1 phosphorylation and S-phase arrest were abrogated by caffeine. However, low doses of BPDE elicited Chk1 phosphorylation and S-phase arrest in AT cells (from ataxia telangiectasia patients), demonstrating that ATM is dispensable for S-phase checkpoint responses to this genotoxin. BPDE-induced Chk1 phosphorylation and S-phase arrest were abrogated by caffeine treatment in AT cells, suggesting that a caffeine-sensitive kinase other than ATM is an important mediator of responses to BPDE-adducted DNA. Overall, our data demonstrate the existence of a caffeine-sensitive, Chk1-mediated, S-phase checkpoint that is operational in response to BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

Synthesis and Electrochemistry of Tin(IV) Octaethylcorroles, (OEC)Sn(C(6)H(5)) and (OEC)SnCl.

Two Sn(IV) corroles were synthesized and electrochemically examined. The investigated compounds are represented as (OEC)Sn(C(6)H(5)) and (OEC)SnCl, where OEC = trianion of 2,3,7,8,12,13,17,18-octaethylcorrole. (OEC)Sn(C(6)H(5)) represents the first example of a sigma-bonded metallocorrole which does not undergo a metal-centered electrode reaction. Both compounds undergo three reversible one-electron oxidations, all of which occur at the conjugated macrocycle. The reduction of (OEC)SnCl involves an overall two electrons, with the product being spectroscopically identified as a Sn(II) corrole after bulk electrolysis of the starting compound. (OEC)Sn(C(6)H(5)) is reversibly reduced by a single electron to give a Sn(IV) corrole pi-anion radical. The electrochemically measured HOMO-LUMO gap (defined as the absolute potential difference between the first-ring centered reduction and first ring-centered oxidation) is equal to 2.25 V in benzonitrile, a value which closely approximates what is observed for porphyrins containing octaethylporphyrin (OEP) or tetraphenylporphyrin (TPP) macrocycles. An X-ray crystallographic analysis for the molecular structure of (OEC)Sn(C(6)H(5)) is also presented: monoclinic, P2(1)/n, with a = 13.235(4) Å, b = 14.502(4) Å, c = 18.387(5) Å, beta = 95.45(2) degrees, Z = 4, R = 0.0619.

Journal Article↗
PubMed1996

Synthesis, Characterization, and Spectroelectrochemistry of Cobalt Porphyrins Containing Axially Bound Nitric Oxide.

Several cobalt nitrosyl porphyrins of the form (T(p/m-X)PP)Co(NO) (p/m-X = p-OCH(3) (1), p-CH(3) (2), m-CH(3) (3), p-H (4), m-OCH(3) (5), p-OCF(3) (6), p-CF(3) (7), p-CN (8)) have been synthesized in 30-85% yields by reaction of the precursor cobalt porphyrin with nitric oxide. Compounds 1-7 were also prepared by reaction of the precursor cobalt porphyrin with nitrosonium tetrafluoroborate followed by reduction with cobaltocene. Compounds 1-8 have been characterized by elemental analysis, IR and (1)H NMR spectroscopy, mass spectrometry, and UV-vis spectrophotometry. They are diamagnetic and display nu(NO) bands in CH(2)Cl(2) between 1681 and 1695 cm(-)(1). The molecular structure of 1, determined by a single-crystal X-ray crystallographic analysis, reveals a Co-N-O angle of 119.6(4) degrees. Crystals of 1 are monoclinic, P2/c, with a = 15.052(1) Å, b = 9.390(1) Å, c = 16.274(2) Å, beta = 111.04(1) degrees, V = 2146.8(4) Å(3), Z = 2, T = 228(2) K, D(calcd) = 1.271 g cm(-)(3), and final R1 = 0.0599 (wR2 = 0.1567, GOF = 1.054) for 3330 "observed" reflections with I >/= 2sigma(I). Cyclic voltammetry studies in CH(2)Cl(2) reveal that compounds 1-7 undergo two reversible oxidations and two reversible reductions at low temperature. This is not the case for compound 8, which undergoes two reversible reductions but an irreversible oxidation due to adsorption of the oxidized product onto the electrode surface. Combined electrochemistry-infrared studies demonstrate that each of the compounds 1-7 undergoes a first oxidation at the porphyrin pi ring system and a first reduction at either the metal center or the nitrosyl axial ligand. The formulation for the singly oxidized products of compounds 1-7 as porphyrin pi-cation radicals was confirmed by the presence of bands in the 1289-1294 cm(-)(1) region (for compounds 1-5), which are diagnostic IR bands for generation of tetraarylporphyrin pi-cation radicals.

Journal Article↗